Structural characterization of the oxygen reactive heamoglobins from the clam Lucina pectinata
Estremera-Andújar, Rafael A.
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Hemoglobins comprise a challenging area of study within biochemical research. They play an important physiological role in organisms, like oxygen transport and storage. Three hemoglobins were discovered in the ctenidia tissue of the Lucina pectinata bivalve mollusk. Hemoglobin I (HbI) is classified as sulfide reactive, while hemoglobin II (HbII), hemoglobin III (HbIII), and the HbII-III complex are unreactive towards H2S. The functional aspects of these oxygen carrier proteins can be explained by a series of contributing factors. The distal heme pocket composition of the heterodimer of HbII-III complex and the homodimer of HbIII and HbII contains TyrB10 and GlnE7, which are common in other hemoglobins of non-vertebrate organisms. These residues contribute to the ligand stabilization and dissociation by a hydrogen bond network with the heme-ligand moiety. The heme pocket size, the hydrogen bonding network, and the orientation of the heme-oxygen complex of the HbII-III complex and that of HbII act together to confer stability to the protein. At pH 7, the crystallographic structure of HbII (2OLP) shows a dimer, where oxygen is tightly anchored to the heme group through hydrogen bonds with TyrB10 and GlnE7. At pH 5, the crystallographic structure of HbII-III complex (3PT7) shows a heterodimeric structure with the same behavior of the homodimer HbII. The mechanism used by these hemoglobins, their function, ligand selection, and stabilization remains partially unknown. This study contributes to the better understanding of this mechanism. Crystals of the Oxy complex of HbII-III were grown at different pH values (4 to 8) using the capillary counterdiffusion (CCD) technique to elucidate the crystallographic structure and to determine conformational changes of the conformations of the TyrB10 and GlnE7 residues; as well as the structural scenarios induced by pH changes. Good prismatic ruby-red crystals were obtained at all pH values with precipitant agents, such as sodium formate and ammonium sulfate. The crystallographic structures of the Oxy heterodimers complexes of HbIIIII have been compared with the Oxy homodimers complexes of HbII, at pH 4 to 8, to determine structural changes, differences in the distal site, the peripheral side groups, and the planarity of the heme pocket. The HbIII component was isolated and purified from the complex of HbII-III with a new protocol. The purity of the HbIII component was confirmed with MS/MS spectrometry. Crystallization conditions for the Oxy and Cyano complexes for HbIII were obtained. X-ray data sets were acquired using synchrotron radiation to a maximum resolution of 2.65, 2.24, 1.86 and 1.85 Å for pH 4, 6, 7 and pH 8, respectively. All obtained crystals were isomorphous, belonging to the P42212 space group. The crystallographic data suggests that pH is a driving force for a conformational change in the HbII-III complex protein structure, specifically in the distal region of the heme group. These changes can promote the rearrangement of the TyrB10 and GlnE11 residues, with or without a hydrogen bond network, with the oxygen ligand in the heme pocket of HbII-III complex at pH values mentioned above. The results indicate a conformational equilibrium between an open and a closed conformational model at the heme pocket for the Oxy complex of HbII-III.